Successful excystation of sporulated Eimeria spp. oocysts is an important step to acquire large numbers of viable sporozoites for molecular, biochemical, immunological and in vitro experiments for detailed studies on complex host cell-parasite interactions. An improved method for excystation of sporulated oocysts and collection of infective E. bovis- and E. arloingi-sporozoites is here described. Eimeria spp. oocysts were treated for at least 20 h with sterile 0.02 M L-cysteine HCl/0.2 M NaHCO3 solution at 37 °C in 100% CO2 atmosphere. The last oocyst treatment was performed with a 0.4% trypsin 8% sterile bovine bile excystation solution, which disrupted oocyst walls with consequent activation of sporozoites within oocyst circumplasm, thereby releasing up to 90% of sporozoites in approximately 2 h of incubation (37 °C) with a 13 (oocystssporozoites) ratio. Free-released sporozoites were filtered in order to remove rests of oocysts, sporocysts and non-sporulated oocysts. Furthermore, live cell imaging 3D holotomographic microscopy (Nanolive®) analysis allowed visualization of differing sporozoite egress strategies. Sporozoites of both species were up to 99% viable, highly motile, capable of active host cell invasion and further development into trophozoite- as well as macroment-development in primary bovine umbilical vein endothelial cells (BUVEC). Sporozoites obtained by this new excystation protocol were cleaner at the time point of exposure of BUVEC monolayers and thus benefiting from the non-activation status of these highly immunocompetent cells through debris. Alongside, this protocol improved former described methods by being is less expensive, faster, accessible for all labs with minimum equipment, and without requirement of neither expensive buffer solutions nor sophisticated instruments such as ultracentrifuges. Vasa and dazl genes have been reported to play pivotal roles in germ cell development and differentiation both in vertebrates and invertebrates; however, little is known about their functions in germ cell differentiation during gametogenesis and sex reversal in hermaphroditic fish. In the present study, vasa (Ecvasa) and dazl (Ecdazl) cDNA were cloned from orange-spotted grouper (Epinephelus coioides). Oxaliplatin The full-length cDNA sequences of Ecvasa and Ecdazl were 2162 and 2101 bp, and encoded 646 and 214 amino acid residues, respectively. Reversal transcription PCR showed that Ecvasa and Ecdazl mRNA were highly expressed in the gonads. Further, in situ hybridization revealed that Ecvasa and Ecdazl RNA were dynamically expressed in germ cells at different stages during oogenesis, sex reversal, and spermatogenesis in orange-spotted grouper. Intriguingly, the signals for Ecvasa and Ecdazl mRNA became weaker in oocytes of ovo-testes gonads, indicating that the expression of germ cell genes could be suppressed in oocytes during sex reversal in the orange-spotted grouper. Our study is the first time to describe the expression profiles of vasa and dazl mRNA in germ cells during gametogenesis and sex reversal in the orange-spotted grouper. These findings will provide new insights into understanding the mechanisms through which vasa and dazl regulate germ cell differentiation in hermaphrodite fish species. V.Accurate assessment of the HER2 expression is an essential issue for predicting response to anti-HER2 therapy in breast cancer patients. The aim of this study was to evaluate 99mTc-HYNIC-(Ser)3-LTVPWY (99mTc-HYNIC-LY) peptide as a novel HER2-targeted radiolabeled peptide in healthy mice to examine the applicability of this imaging agent in a first-in-human clinical trial. To this end, pharmacokinetic and dosimetry studies were performed according to the ICH guideline M3 (R2) with 99mTc-HYNIC-LY. To estimate the radiation-absorbed doses in humans, the accumulated activity in each mouse organ was calculated based on biodistribution data. In addition, toxicology assessment was performed based on mortality events, body weights, and serum biochemical, hematological, and histopathological assays. The pharmacokinetic study showed rapid blood clearance. Based on the results of biodistribution study, the highest radioactivity was observed in the kidneys. The projected absorbed doses to the kidneys, liver, lungs, stomach, and spleen were obtained as 1.70E-02, 1.42E-02, 1.02E-02, 8.62E-03, and 8.34E-03 mSv/MBq, respectively. The results also revealed that serum biochemical and hematological parameters were in the normal range. No significant morphologic alterations were observed in the liver, kidneys, and spleen tissues. Consequently, the results were indicative of the suitability of 99mTc-HYNIC-LY peptide for advancement to a first-in-human clinical trial. In human risk assessment, time extrapolation factors (EFs) account for differences in exposure duration of experimental studies. We calculated EFs based on N(L)OEL (no (lowest) observed effect level) ratios, dividing shorter-term by longer-term values. The ‘oral’ datasets comprised 302 EFs (subacute-subchronic) and 1059 EFs (subchronic-chronic). The ‘inhalation’ datasets contained 67 EFs (subacute-subchronic) and 226 EFs (subchronic-chronic). The experimental EF distribution oralsubchronic-chronic showed that study parameters like deviation in dose selection and spacing influence mainly the data variance. Exclusion of these influences led to a dataset representing more realistically the difference of N(L)OELs with prolonged treatment. This dataset showed a GM of 1.5, indicating that the impact of a longer treatment period on the study N(L)OEL is on average not high. A factor of 1.5 seemed to be also sufficiently conservative for subacute-subchronic and subchronic-chronic extrapolation (inhalation or oral exposure). EFs for groups of similar compounds did not differ, but for compounds with low and high NOEL values. Relatively toxic compounds (GM 1) might thus not require time extrapolation. Within and between chemical variance was analysed in the dataset oralsubchronic-chronic (GSD 4.8). The variance between chemicals should be considered within extrapolation by selecting an appropriate percentile for which a chemical variance factor is suggested. In risk assessment, often a combination of EFs is required. Our analysis indicates that such a combination will result in an accumulation of non-toxicological variance and therefore unrealistically high EFs. Further evaluations are needed to identify appropriate chemical variance factors for these situations.